Advances in the areas of the use of recombinant viral vectors for gene therapy and DNA vaccination applications have created a need for large scale manufacture and purification of clinical-grade virus. One such family of viruses are the adenoviruses. The adenoviruses are grouped within the family Adenoviridae, which are split into the genus Aviadenovirus (birds) and Mastadenovirus (human, simian, bovine, equine, porcine, ovine, canine and opossum). A review of the family Adenoviridae can be found in Fundamental Biology, 3rd Ed., Fields, B. N., Knipe, D. M., and Howley, P. M., Ed., at Chapter 30, pp. 979-1016 (1996), which is hereby incorporated by reference. Of specific interest in gene vaccination and/or gene therapy applications is the use of a replication incompetent adenovirus, crippled by E1 or further deletions, including “gutless” adenovirus vectors. The adenovirus genome is generally associated with benign pathologies in humans, and the genomic organization of the virus has been well studied since its discovery in the early 1950s. In addition, the genome is amenable to manipulation, depending on the strategy utilized to construct the respective vector. A replication-incompetent virus (such as an E1/E3 deleted Ad5gag vector expressing a HIV gag transgene, as exemplified herein) requires a cell line which complements the deletions. Any such cell line may be used to generate recombinant virus vectors, with preferred, but not limiting, cell lines including 293 cells and PER.C6™ cells. To this end, numerous 1st generation recombinant adenovirus vectors have been described in the literature (e.g., see Bett, et al., 1994, Proc. Natl. Acad. Sci. 91:8802-8806; WO 01/02607 and WO 02/22080). “Gutless” adenoviral vectors are a 2nd generation adenoviral vector generally devoid of viral protein-coding sequences, frequently with viral proteins supplemented in trans by a helper virus (often an E1-deleted adenovirus) grown with the helper-dependent (HD) adenovector in a packaging cell line (e.g., PER.C6™). Absent viral proteins, these viral vectors can, in the alternative, be supplemented in trans by a cell line and/or “helper virus” capable of expressing the structural and functional adenoviral proteins necessary for successful replication, packaging and rescue. In view of the increased popularity of these viral vectors and the ultimate need to prepare commercial scale quantities of either a viral vector based vaccine or gene therapy vehicle, it has become essential to develop more efficient qualitative and quantitative methodology for production of commercial grade recombinant adenovirus vectors.
It has been shown that temperature is an important process parameter for both cell growth and virus production. The physiological temperature of 37° C. has been shown to be optimal for growth of a majority of mammalian cell lines. Temperatures below 37° C. historically are shown to reduce cell growth rate, overall cell metabolism, and specific product formation in mammalian cells (see, e.g., Moore, et al., 1997, Cytotechnology 23:47-54; Chuppa, et al., 1997, Biotechnol Bioeng. 55:328-338). The optimal temperature for virus production depends on the virus strain and the host cell line, but has most often been found to be below 37° C., including 34° C. for herpes simplex virus (HSV) production in FL cell culture (Hoggan and Roizman, 1959, Virology 8:508-524), 32 to 34° C. for myxoma virus (Ross and Sanders, 1979, J. Gen. Virol. 43:213-216), and 35° C. for foot-and-mouth disease virus in suspension BHK 21 cell cultures (Capstick et al., 1967, J. Hyg. Camb. 65:273-280), and 32° C. for cold adapted influenza viruses in MDCK cells. Temperatures above 37° C. are generally not suitable or even non-permissive for virus replication (Schweitzer-Thumann et al., 1994, Res. Virol. 145:163-170). For highly heat-labile viruses such as retrovirus, virus productivity can be significantly increased by shifting the culture temperature down from 37° C. (during cell growth) to 32° C. for virus production, primarily due to increased virus stability at a lower temperature (McTaggart and Al-Rubeai, 2000, Biotechnol. Prog. 16:859-865).
Despite these reports, there remains a need for the development of a large scale process for virus production from cell culture which address both quantitative and qualitative issues that are imposed upon a commercialized viral-based vaccine and/or gene therapy product. The present invention addresses and meets these needs by disclosing an optimized cell culture and virus production process which defines optimal temperature ranges, resulting in an improved virus productivity as well as elimination of intra-batch productivity variations.